Engineering Machinery Molecules to Visualize and Reprogram Immunotherapy Abstract Adoptive immunotherapy has the potential to become a paradigm shifting technology for cancer therapy. Specifically, cell based immunotherapy has demonstrated phenomenal success in clinical trials against various malignancies. Although promising, a high degree of precise control of engineered immunocells in targeting tumor cells is needed before the cell-based therapy can become widely adopted. Synthetic biology is an emerging field with the overall goal to understand and manipulate life processes using an engineering approach, particularly molecular engineering. Naturally occurring molecules and domains can be modulated and integrated to produce regulatory modules and molecular machines with well controlled functions. This will allow the engineering of controllable machinery molecules capable of detecting antigens/biomarkers to activate cellular immuno- responses. Biosensors based on fluorescence resonance energy transfer (FRET) have revolutionized the biomedical research by allowing direct visualization and characterization of molecular activities in live cells. The functionality and efficacy of the machinery molecules as well as their modular components can hence be precisely characterized by FRET biosensors serving as ?digital multimeters? to provide immediate feedbacks for the optimization of the sophisticated machinery molecules. We aim to engineer integrated machinery molecules which can provide a surveillance of the intracellular space, visualizing the spatiotemporal patterns of specific biochemical events and automatically triggering molecular actions to guide immuno-cell functions. We have adopted a modular assembly approach to develop a machinery molecule, specifically for the sensing of intracellular phosphorylation and consequent activation of a tyrosine phosphatase (PTP) Shp2, which plays a critical role in various pathophysiological processes. We have further integrated this machinery molecule to the ?don't eat me? CD47 receptor SIRP? in macrophages such that the engagement of SIRP? and its activation of naturally negative signals will be rewired to turn on a positive Shp2 action to activate the engineered macrophages and facilitate phagocytosis initiated by an anti-gen-targeting antibody and its interaction with Fc? receptors. In this proposal, we plan to apply this strategy to re-engineer macrophages for the eradication of tumors. We choose colon cancer in which cells express a high level of CD47 as our first proof-of-concept target. Two specific aims are accordingly proposed: Specific Aim 1. Characterize the phagocytic efficiency and its associated FRET signals of re-engineered macrophages against colon cancer cells. Specific Aim 2. Examine the efficiency of re-engineered macrophages in eradicating the colon tumors in nude and immunocompetent mouse models. Our platform is designed to be highly modular, with each functional module readily switchable to rewire the molecular network. The platform allows new mode of tumor eradication, with a switchable antibody interface for the eradiation, in principle, any type of tumors. Our engineered macrophage can also be combined with traditional radiotherapy and chemotherapy approaches as well as the immunotherapy antibody methods mitigating the immuno-inhibitory PD-1 and CTLA-4 signals. Vitamin D analog in modulating stromal cells, and the hyaluronic acid (HA) signaling inhibitor PEGPH20 in promoting extracellular matrix (ECM) depletion and tumor vascularity can modulate tumor microenvironment. These existing methods can also be combined together with our engineered macrophage approach to improve the therapeutic efficiency of tumor immunotherapy. Therefore, the success of the proposed approach will revolutionize the ability to perform cell-based immunotherapy and highlight the translational power in bridging the fundamental molecular engineering to clinical medicine.